Insight into a wide range of clinical situations may be gained from salivary analysis (See e.g., Mandel, I. D., “Salivary Diagnosis: Promises, Promises,” in Saliva as a Diagnostic Fluid, Malamud et al., eds., Ann. NY Acad. Sciences 694: 1-10 (1993)). The earliest sialochemical studies on oral fluids examined saliva for specific components that would be diagnostic for various systemic conditions, including gout and rheumatism. (Atkinson, et al., “Guidelines for Saliva Nomenclature and Collection,” in Saliva as a Diagnostic Fluid, Ann. NY Acad. Sciences 694: xi-xii (1993)). A recent example utilizes salivary acetaminophen concentration to assess the gastric emptying rate of liquids. A number of studies report using saliva specimens for monitoring pharmaceuticals and chemicals, including taxol (Svojanovsky, et al., J. Pharm. Biomed. Anal., 20:549-555 (1999)); caffeine (Akinyinka, et al., Eur. J. Clin. Pharmacol., 56:159-165 (2000)); and the protease inhibitor, indinavir (Wintergerst, et al., Antimicrobial Agents and Chemotherapy, 44:2572-2574 (2000)).
In general, the analysis of saliva for diagnostic purposes has been directed towards evaluating systemic disease (e.g., Sjogren's syndrome, cystic fibrosis, HIV infection, etc.), or as a means of determining systemic levels of therapeutic drugs such as steroids (Ferguson, J. Dent. Res. 66(2): 420-424 (1987)). There have also been many attempts to measure other factors in saliva and then relate them to oral diseases. For example, salivary analysis has also been used to diagnose periodontal disease (U.S. Pat. No. 6,063,588 to Lamster; U.S. Pat. No. 5,376,532 to Singer, Jr.; U.S. Pat. Nos. 5,866,432 and 5,736,341 to Sorsa, et al.; and U.S. Pat. No. 5,756,361 to Winterbottom, et al.). However, the vast majority of studies have not been able to relate factors in saliva with other common oral diseases such as dental caries. These studies reported values for pH, various ions, macromolecules, and flow rate, but found little evidence of a correlation. The few studies that have shown a small amount of correlation were found not to correlate with other studies. Thus, there remains a need for a saliva-based test for predicting the risk of oral diseases and associated diseases that is simple and accurate.
I. Oral Diseases and Associated Diseases
The most common oral diseases are dental caries and the periodontal diseases. Individuals are vulnerable to dental caries throughout life, affecting 85 percent of adults aged 18 and older. Periodontal diseases are most often seen in maturity, with the majority of adults experiencing some signs and symptoms by the mid-30s. Certain rare forms of periodontal disease affect young people. Oral disorders also include certain mucosal infections, as well as oral and pharyngeal cancers and precancerous lesions.
A. Dental Caries
Caries is a unique multifactorial infectious disease (Lenander-Lumikari, et al., Adv. Dent. Res. 14:4047 (December 2000)). Dental caries affects teeth at all levels and can cause extensive crown mutilations, bacterial disorders of the periapical tissues, or even loss of the affected dental elements. Clinically, the disease is characterized by demineralization of the dental enamel and of the dentin in various stages of progress, until it affects the pulp space. When the lesion passes beyond the enamel-dentin border, a phlogistic reaction of the pulp tissues is constantly observed, with the formation of reaction dentin in some cases. Approximately 50% of adult individuals have at least four caries-related lesions that are treated and require treatment, and approximately 30% of adult individuals have over 50% of their teeth affected by caries (U.S. Pat. No. 5,830,489 to Valenti, et al.).
The bacterium Streptococcus mutans, or S. mutans, is known to be a prime etiologic agent for the initiation and progression of human dental caries, or cavities. S. mutans is one of the primary factors in acid dissolution of the apatite (mineral) component of the enamel then the dentin, or of the cementum then the dentin (Tanzer, J. M., Inter. J. Oral Biol. 22:205-214 (1997)). A strong correlation between the proportion of S. mutans in dental plaque or in saliva relative to other bacterial species and the presence or risk of future outbreaks of dental caries has been documented (Tanzer, J. M., supra). Therefore, S. mutans in plaque or saliva may serve as an index for both caries activity state and caries risk or susceptibility. These indices play an increasingly important role in the diagnosis and treatment of dental caries (Hume, W. R., J. Dent. Educ. 57:439-443 (1993)).
Present techniques for detecting and quantitatively determining S. mutans include bacterial culture with selective media using either broth or agar plate systems, and polymerase chain reaction techniques (Ellen, R. P., Oral Sci. Rev. 8:3-23 (1976); Igarashi, et al., Oral Microbiol. and Immunol. 11:294-298 (1996); U.S. Pat. No. 5,374,538 to Bratthall; U.S. Pat. No. 4,692,407 to Jordan, et al.). However, each of these methods require significant time. (on the order of days), well trained personnel and sophisticated equipment to perform. Consequently, existing techniques are relatively expensive and time consuming. Moreover, the use of the titer of S. mutans in the oral cavity as a predictor of caries risk is consistently significant only within the first two years of age.
Human dental caries may also be detected by changes in translucency, color, hardness or X-ray density of teeth. However, these technologies have limitations both in specificity and reproducibility. Furthermore, they do not show whether or not the disease is active at a single time point (U.S. Pat. No. 6,231,857 to Shi, et al.).
B. Periodontal Diseases
Like dental caries, the periodontal diseases are infections caused by bacteria in the biofilm (dental plaque) that forms on oral surfaces. The basic division in the periodontal diseases is between gingivitis, which affects the gums, and periodontitis, which may involve all of the soft tissue and bone supporting the teeth. Gingivitis and milder forms of periodontitis are common in adults. The percentage of individuals with moderate to severe periodontitis, in which the destruction of supporting tissue may cause the tooth to loosen and fall out, increases with age.
1. Gingivitis
Gingivitis is an inflammation of the gums characterized by a change in color from normal pink to red, with swelling, bleeding, and often sensitivity and tenderness. These changes result from an accumulation of biofilm along the gingival margins and the immune system's inflammatory response to the release of destructive bacterial products. The early changes of gingivitis are reversible with thorough toothbrushing and flossing to reduce plaque. Without adequate oral hygiene, however, these early changes can become more severe, with infiltration of inflammatory cells and establishment of a chronic infection. Biofilm on tooth surfaces opposite the openings of the salivary glands often mineralizes to form calculus or tartar, which is covered by unmineralized biofilm—a combination that may exacerbate local inflammatory responses (Mandel, J. Am. Dent. Assoc., 126:573-80 (1995)). A gingival infection can persist for months or years, yet never progress to periodontitis.
Gingival inflammation does not appear until the biofilm changes from one composed largely of gram-positive streptococci (which can live with or without oxygen) to one containing gram-negative anaerobes (which cannot live in the presence of oxygen). Numerous attempts have been made to pinpoint which microorganisms in the supragingival (above the gum line) plaque are the culprits in gingivitis. Frequently mentioned organisms include Fusobacterium nucleatum, Veillonella parvula, and species of Campylobacter and Treponema. 
Gingival inflammation may be influenced by steroid hormones, occurring as puberty gingivitis, pregnancy gingivitis, and gingivitis associated with birth control medication or steroid therapy. The presence of steroid hormones in tissues adjacent to biofilm apparently encourages the growth of certain bacteria and triggers an exaggerated response to biofilm accumulation (Caton, “Periodontal diagnosis and diagnostic aids,” in Proceedings of the World Workshop in Clinical Periodontics, American Academy of Periodontology, pp. I-1-22, Princeton, N.J. (1989)). Certain prescription drugs may also lead to gingival overgrowth and inflammation. These include the antiepileptic drug phenytoin (DILANTIN®), cyclosporin, and various calcium channel blockers used in heart disease.
2. Adult Periodontitis
The most common form of adult periodontitis is described as general and moderately progressing. A second form is described as rapidly progressing and severe, and is often resistant to treatment. The moderately progressive adult form is characterized by a gradual loss of attachment of the periodontal ligament to the gingiva and bone, along with loss of the supporting bone. It is most often accompanied by gingivitis (Genco, “Classification of clinical and radiographic features of periodontal diseases,” in Contemporary Periodontics, Genco et al., eds., pp. 63-81, (1990)). It is not necessarily preceded by gingivitis, but the gingivitis-related biofilm often seeds the subgingival plaque. The destruction of periodontal ligament and bone results in the formation of a pocket between the tooth and adjacent tissues, which harbors subgingival plaque. The calculus formed in the pocket by inflammatory fluids and minerals in adjacent tissues is especially damaging (Mandel and Gaffar, J. Chin. Periodontol., 13: 249-57 (1986)).
The severity of periodontal disease is determined through a series of measurements, including the extent of gingival inflammation and bleeding, the probing depth of the pocket to the point of resistance, the clinical attachment loss of the periodontal ligament measured from a fixed point on the tooth (usually the cemento-enamel junction), and the loss of adjacent alveolar bone as measured by x-ray (Genco, J. Periodontol., 67(10 Suppl.): 1041-9 (1996)). Severity is determined by the rate of disease progression over time and the response of the tissues to treatment. Adult periodontitis often begins in adolescence but is usually not clinically significant until the mid-30s. Prevalence and severity increase but do not accelerate with age (Beck, Ann. Periodontol., 7(1): 322-57 (1996)).
3. Early-Onset Periodontitis
The forms of periodontitis occurring in adolescents and young adults generally involve defects in neutrophil function (Van Dyke et al., Infect. Immun. 27(1):4-24-3-1-124-32 (1980)). Localized juvenile periodontitis (LJP) mainly affects the first molar and incisor teeth of teenagers and young adults, with rapid destruction of bone but also no telltale signs of inflammation and very little supragingival plaque or calculus. Actinobacillus actinomycetemcomitans has been isolated at 90 to 100 percent of diseased sites in these patients, but is absent or appears in very low frequency in healthy or minimally diseased sites (Socransky and Haffajee, J. Periodontol., 63(4 Suppl.):332-31 (1992)). It is possible that the bacteria are transmitted among family members through oral contacts such as kissing or sharing utensils, because the same bacterial strain appears in affected family members. However, evidence of a neutrophil defect argues for a genetic component. Another organism frequently associated with LJP is Capnocytophaga ochracea. Neither of these bacteria dominate in the generalized adult form of the disease, where Porphyromonas gingivalis is considered of greatest significance (Schenkein and Van Dyke, Periodontol., 6:7-25 (1994)).
Prepubertal periodontitis is rare and may be either general or localized. The generalized form begins with the eruption of the primary teeth and proceeds to involve the permanent teeth. There is severe inflammation, rapid bone loss, tooth mobility, and tooth loss. The localized form of the disease is less aggressive, affecting only some primary teeth. The infection involves many of the organisms associated with periodontitis, but the mix can differ somewhat, with Actinobacillus actinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, and several species of Capnocytophaga implicated (Caton, supra). Defects in neutrophil function in both forms of the disease may explain why patients are highly susceptible to other infections as well (Suzuki, Dent. Clin. North Am., 32(2): 195-216 (1988)).
C. Other Diseases Associated With Oral Disorders
Chronic obstructive pulmonary disease, characterized by obstruction of airflow due to chronic bronchitis or emphysema and by recurrent episodes of respiratory infection, has been associated with poor oral health status (Hayes et al., Ann. Periodontol. 3(1):257-61 (1998); Scannapieco et al., Ann. Periondontol., 3(1):251-6 (1998)). A positive relationship between periodontal disease and bacterial pneumonia has also been shown (Scannapieco and Mylotte, J. Periodontol., 67(10 Suppl.): 1114-22 (1996)).
Recent studies have also underscored the association of oral infections with certain medically important conditions. Increasing data implicate periodontal disease as a risk factor for cardiovascular diseases such as heart attack and stroke (See e.g., U.S. Pat. No. 6,130,042 to Diehl, et al.; J. Beck, et al., J. Periodontol., 67:1123-37 (1996)). Epidemiologic studies indicate that, even after accounting for other known risk factors for cardiovascular disease, the relative risk attributable to periodontal infections is significant. Secondly, recent studies have shown that mothers with periodontitis are at greater risk for having low weight babies than those without periodontitis (Offenbacher et al., J. Periodontol., 67:1103-13 (1996)).
There is also growing acceptance that diabetes is associated with increased occurrence and progression of periodontitis-so much so that periodontitis has been called the “sixth complication of diabetes” (Loe, Diabetes Care, 16(1): 329-34 (1993)). The risk is independent of whether the diabetes is type 1 or type 2. Type 1 diabetes is the condition in which the pancreas produces little or no insulin. It usually begins in childhood or adolescence. In type 2 diabetes, secretion and utilization of insulin are impaired; onset is typically after age 30. Together, these two types of diabetes affect an estimated 15.7 million people in the United States and represent the seventh leading cause of death (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Diabetes statistics. NIH Pub. No. 99-3892 (1999)). The goal of diabetic care is to lower blood glucose levels to recommended levels. Some investigators have reported a two-way connection between diabetes and periodontal disease, proposing that not only are diabetic patients more susceptible to periodontal disease, but the presence of periodontal disease affects glycemic control.
D. Oral Disease and Adverse Pregnancy Outcomes
Preterm birth and low birth weight are considered the leading perinatal problems in the United States (Gibbs et al., Am. J. Obstet. Gynecol., 166(5): 1515-28 (1992)). Although infant mortality rates have decreased substantially over the past generation, the incidence of low birth weight (just under 300,000 cases in 1995) has not shown a comparable decline (Institute of Medicine, Committee to Study the Prevention of Low Birth Weight, Division of Health. Promotion and Disease Progression. Preventing low birth weight. Washington: National Academy Press (1985)). Over 60 percent of the mortality of infants without structural or chromosomal congenital defects may be attributed to low birth weight (Shapiro, et al., Am. J. Obstet. Gynecol., 136(3): 363-73 (1980)).
Oral disease may contribute to adverse outcomes of pregnancy as a consequence of a chronic oral inflammatory bacterial infection. For example, toxins or other products generated by periodontal bacteria in the mother can reach the general circulation, cross the placenta, and harm the fetus. In addition, the response of the maternal immune system to the infection elicits the continued release of inflammatory mediators, growth factors, and other potent cytokines, which may directly or indirectly interfere with fetal growth and delivery.
Evidence of increased rates of amniotic fluid infection, chorioamnion infection, and histologic chorioamnionitis supports an association between preterm birth, low birth weight, and general infection during pregnancy. It is noteworthy that the largest proportion of such infections occurred during the pregnancies of the most premature births (Hillier, et al., N. Engl. J. Med. 319(15): 972-8 (1988); Hillier, et al., N. Engl. J. Med., 333(26):1737-42 (1995)). The biological mechanisms involve bacteria-induced activation of cell-mediated immunity leading to cytokine production and the synthesis and release of prostaglandins, which may trigger preterm labor (Hillier, et al., supra). Elevated levels of prostaglandin as well as cytokines-(interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) have been found in the amniotic fluid of patients in preterm labor with amniotic fluid infection (Romero, et al., Am. J. Obstet. Gynecol., 186(6 Pt 1):1654-64 (1993)), compared with levels in patients with preterm labor without infection.
E. Mucosal Infections
The mucosal lining of the mouth is subject to a variety of infections and conditions, ranging from benign canker sores to often fatal cancers.
Oral Candidiasis
Chronic hyperplastic candidiasis is a red or white lesion that may be flat or slightly elevated and may adhere to soft or hard tissue surfaces, including dental appliances. It is caused by species of Candida, especially Candida albicans, the most common fungal pathogen isolated from the oral cavity. Normally, the fungi are present in relatively low numbers in up to 65 percent of healthy children and adults and cause no harm (McCullough, et al., Int. J. Oral Maxillofac. Surg., 25:136-44 (1996)).
The most common form of oral candidiasis is denture stomatitis. It occurs when tissues are traumatized by continued wearing of ill-fitting or inadequately cleaned dental appliances and is described as chronic erythematous candidiasis. Another form, candidal angular cheilosis, occurs in the folds at the angles of the mouth and is closely associated with denture sore mouth (Tyldesley and Field, Oral Medicine, 4th ed., Oxford University Press (1995)). Other common forms of Candida infection are pseudomembranous candidiasis (thrush), which may affect any of the mucosal surfaces, and acute erythematous candidiasis, a red and markedly painful variant commonly seen in AIDS patients.
In most cases, Candida infection may be controlled with antifungal medications used locally or systemically. Control is difficult, however, in patients with immune dysfunction, as in AIDS, or other chronic debilitating diseases. Often the organisms become resistant to standard therapy, and aggressive approaches are necessary (Tyldesley and Field, supra). The spread of oral candidiasis to the esophagus or lungs may be life-threatening and is one of the criteria used to define frank AIDS (Samaranayake and Holmstrup, J. Oral Pathol. Medi., 18:554-64 (1989)).
F. Herpes Simplex Virus Infections
In any given year, about one-half-million Americans will experience their first encounter with the herpes simplex virus type 1 (HSV-1), the cause of cold sores. That first encounter usually occurs in the oral region and can be so mild as to go unnoticed. But in some people, particularly young children and young adults, infection may take the form of primary herpetic stomatitis, with symptoms of malaise, muscle aches, sore throat, and enlarged and tender lymph nodes, prior to the appearance of the familiar cold sore blisters. These blisters usually show up on the lips, but any of the mucosal surfaces may be affected. Bright-red ulcerated areas and marked gingivitis can also be seen (Tyldesley and Field, supra).
Herpes viruses also cause genital infections, which are transmitted sexually. Both HSV-1 and HSV-2 have been found in oral and genital infections, with HSV-1 predominating in oral areas and HSV-2 in genital areas (Wheeler, J. Am. Acad. Dermatol., 18(1 Pt. 2): 163-8 (1988)). Herpes viruses have also been implicated as cofactors in the development of oral cancers. Crowded living conditions may result in greater contact with infected individuals, which aids in transmission of HSV (Whitley, Pathol. Biol., 40(7): 729-34 (1992)).
G. Oral Human Papillomavirus Infections.
There are more than 100 recognized strains of oral human papillomavirus (HPV), a member of the papovavirus family, implicated in a variety of oral lesions (Regezi and Sciubba, Oral pathology. Clinical-pathologic correlations, 2nd ed., (1993)). Most common are papillomas (warts) found on or around the lips and in the mouth. HPV is found in 80 percent of these oral squamous papillomas (de Villiers, Biomed Pharmacother., 43:31-6 (1989)). The virus has also been identified in 30 to 40 percent of oral squamous cell carcinomas (Chang, et al., Arch. Dermatol. Res., 282(8):493-497 (1990)) and has been implicated in cervical cancer as well. Whether a cancer or nonmalignant wart develops may depend on which virus is present or on which viral genes are activated. Oral warts are most often found in children, probably as a result of chewing warts on the hands. In adults, sexual transmission from the anogenital region may occur (Franchesi, et al., Cancer Epidemiol. Biomarkers Prev., 5:565-575 (1996)). In general, viral warts spontaneously regress after 1 or 2 years. The immune system normally keeps HPV infections under control, as evidenced by the increased prevalence of HPV-associated lesions in HIV-infected patients and others with immunodeficiency.
H. Recurrent Aphthous Ulcers
Recurrent aphthous ulcers (RAU), also referred to as recurrent aphthous stomatitis, is the technical term for canker sores, the most common and generally mild oral mucosal disease. Between five and twenty-five percent of the general population is affected, with higher numbers in selected groups, such as health professional students (Ferguson, et al, J. Oral Med., 39(4):212-217 (1984); Kleinman, et al., Community Dent. Oral Epidemiol., 5:140-144 (1991)).
The disease takes three clinical forms: RAU minor, RAU major, and herpetiform RAU. The minor form accounts for 70 to 87 percent of cases. The sores are small, discrete, shallow ulcers surrounded by a red halo appearing at the front of the mouth or the tongue. The ulcers, which usually last up to two weeks, are painful and can make eating or speaking difficult. About half of RAU patients experience recurrences every one to three months; as many as thirty percent report continuous recurrences (Bagan et al., J. Oral Pathol. Med., 20:395-7 (1991)).
RAU major accounts for seven to twenty percent of cases and usually appears as one to ten larger coalescent ulcers at a time, which may persist for weeks or months (Bagan, et al., supra). Herpetiform RAU has been reported as occurring in seven to ten percent of RAU cases. The ulcers appear in crops of ten to one hundred at a time, concentrating in the back of the mouth and lasting for seven to fourteen days (Bagan, et al., supra).
RAU can begin in childhood, but the peak period for onset is the second decade (Lehner, Proc. R. Soc. Med., 61:515-24 (1968)). About fifty percent of close relatives of patients with RAU also have the condition (Ship, J. Dent. Res., 44:837-44 (1965)), and a high correlation of RAU has been noted in identical but not fraternal twins. Associations have been found between RAU and specific genetic markers (Scully and Porter, J. Oral Pathol. Med., 18:21-7 (1989)).
RAU has also been associated with hypersensitivities to some foods, food dyes, and food preservatives (Woo and Sonis, J. Am. Dent. Assoc., 127(8):1202-13 (1996)). Nutritional deficiencies-especially in iron, folic acid, various B vitamins, or combinations thereof have also been reported, and improvements noted with suitable dietary supplements (Nolan, et al., J. Oral Pathol. Med., 20:389-91 (1991)).
I. Oral and Pharyngeal Cancers and Precancerous Lesions
Oral cancer is the sixth most common cancer in U.S. males and takes a disproportionate toll on minorities; it now ranks as the fourth most common cancer among African American men. The most common oral sites are on the tongue, the lips, and the floor of the mouth.
Viruses that have been implicated in oral cancer include herpes simplex type 1 and human papillomavirus. Epstein-Barr virus, also a herpes virus, is now accepted as an oncogenic virus responsible for Burkitt's lymphoma, occurring primarily in Africa, and nasopharyngeal carcinoma, occurring primarily in China. HPV is a major etiologic agent in cervical cancer, and has been found in association with oral cancer as well (Sugerman and Shillitoe, Oral Dis., 3:130-47 (1997)). HPV DNA sequences have been found in oral precancerous lesions as well as in squamous cell carcinomas (Syrjanen, et al., J. Oral Pathol., 17(6):273-8 (1988)), and experimental evidence has shown that HPV-16 may be an important cofactor in oral carcinogenesis (Park et al., Oncogene, 10(11: 2145-53 (1995)). Herpes simplex type 1 antibodies were demonstrated in patients with oral cancer, and herpes was found to induce dysplasia (abnormal cellular changes) in the lips of hamsters when combined with the application of tobacco tar condensate.
More recently, human herpes virus 8, a newly identified member of the herpes virus family, has been found in Kaposi's sarcoma, an otherwise rare cancer occurring in patients with AIDS. These tumors often appear initially within the oral cavity (Epstein and Scully, Int. J. Oral Maxillofac. Surg., 21(4):219-26 (1992)). Other uncommon oral malignant tumors, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, may also occur in the mouths of AIDS patients. In addition to viruses, infection with strains of the fungus Candida albicans has been linked to the development of oral cancers via the fungal production of nitrosamines, which are known carcinogens.
J. Associated Autoimmune Disorders
Oral, dental, or craniofacial signs and symptoms play a critical role in autoimmune disorders such as Sjogren's syndrome, and in a number of chronic and disabling pain conditions. Sjogren's syndrome is one of several autoimmune disorders in which the body's own cells and tissues are mistakenly targeted for destruction by the immune system. Like other autoimmune conditions, Sjogren's syndrome is more prevalent among women. The ratio of females to males affected is 9:1, with symptoms usually developing in middle age. There are an estimated one to two million individuals in the United States with Sjogren's syndrome (Talal, Rheum. Dis. Clin. North Am., 18(3):507-15 (1992)).
The disease occurs in two forms. Primary Sjogren's involves the salivary and lacrimal (tear) glands. In secondary Sjogren's the glandular involvement is accompanied by the development of a connective tissue or collagen disease, most often rheumatoid arthritis, lupus erythematosis, scleroderma, or biliary cirrhosis.
The glandular involvement causes a marked reduction in fluid secretion, resulting in xerostomia and xerophthalmia (dry eyes). The constant oral dryness causes difficulty in speaking, chewing, and swallowing; the dry eyes often itch and feel gritty. There is no cure for Sjogren's, and patients often carry eye drops and water bottles or saliva substitutes in an attempt to provide symptomatic relief. Clinically, the reduction in salivary flow changes the bacterial flora, which, in addition to the reduction in salivary protective components, increases the risk of caries and candidiasis (Daniels and Fox, Rheum. Dis. Clin. North Am., 18:571-589 (1992)). Recent studies have indicated that there is a reduction in masticatory function (Dusek, et al. Gerodontology 13(1): 3-6 (1996)) and an increased prevalence of periodontal disease (Najera, et al., Oral Surg. Oral Med. Oral Pathol Oral Radiol. Endol., 83(4)453-7 (1997)). In advanced stages the salivary glands can well because of obstruction and infection or lymphatic infiltration. In both forms of the disease, other systems can eventually become affected. Nasal, laryngeal, and vaginal dryness can occur, as well as abnormalities in internal organs (Oxholm and Asmussen, J. Intern. Med., 239:467-474 (1996)). Patients with Sjogren's syndrome are at some risk of developing diseases such as non-Hodgkin's lymphoma; clinical data indicate that such lymphomas develop in 5 percent of patients with Sjogren's syndrome.
K. HIV and Osteoporosis
The mouth may serve as an early warning system, diagnostic of systemic infectious disease and predictive of its progression, such as with HIV infection. In the case where oral cells and tissues have counterparts in other parts of the body, oral changes may indicate a common pathological process. During routine oral examinations and perhaps in future screening tests, radiographic or magnetic resonance imaging of oral bone may be diagnostic of early osteoporotic changes in the skeleton.
L. HIV Infection
The progressive destruction of the body's immune system by HIV leads to a number of oral lesions, such as oral candidiasis and oral hairy leukoplakia, that have been used not only in diagnosis but also in determining specific stages of HIV infection. Oral candidiasis is rarely seen in previously healthy young adults who have not received prior medical therapy such as cancer chemotherapy or treatment with other immunosuppressive drugs. Oral candidiasis may be the first sign of HIV infection and often occurs as part of the initial phase of infection—the acute HIV syndrome (Tindall, et al., “Primary HIV infection: Clinical, Immunologic, and Serologic Aspects,” in The Medical Management of AIDS, Sande, et al., eds., pp. 105-129; W. B. Saunders, 1995). It tends to increase in prevalence with progression of HIV infection when CD4 lymphocyte counts fall. It also appears to be the most common oral manifestation in pediatric HIV infection (Kline, Pediatrics, 97(3):380-388 (1996) and has been demonstrated to proceed to esophageal candidiasis, a sign of overt AIDS. (Saah et al., Am. J. Epidemiol., 135:1147-1155 (1992)). Both the pseudomembranous and the erythematous forms of candidiasis appear to be important predictors of progression of HIV infection (Klein et al., AIDS, 6(3): 332-333 (1992)).
Like oral candidiasis, oral hairy leukoplakia in HIV-positive persons heralds more rapid progression to AIDS. Oral hairy leukoplakia is an oral lesion first reported in the early days of the AIDS epidemic. Since its discovery, hairy leukoplakia has been found in HIV-negative persons with other forms of immunosuppression, such as organ or bone marrow recipients and those on long-term steroid therapy, and less frequently among immunocompetent persons.
Linear gingival erythema and necrotizing ulcerative periodontitis may be predictive of progression of HIV infection. (Mealey, Ann. Periodontaol., 1:256-321 (1996)). Necrotizing ulcerative periodontitis, a more serious periodontal condition observed in HV-infected persons, is a good predictor of CD4+ cell counts of under 200 per cubic millimeter. In addition, the numerous ulcerative and nonulcerative conditions that affect the oral cavity may affect the biologic activity of HIV and are affected by its treatments (Mealey, supra).
M. Osteoporosis and Oral Bone Loss
Osteoporosis, a degenerative disease characterized by the loss of bone mineral and associated structural changes, has long been suspected as a risk factor for oral bone loss. In addition, measures of oral bone loss have been proposed as potential screening tests for osteoporosis. Osteoporosis affects over 20 million people in the United States, most of whom are women, and results in nearly 2 million fractures per year (National Institute of Arthritis, Musculoskeletal and Skin Diseases 2000). The disease is more prevalent in white and Asian American women than in black women. Oral bone loss has been reported to be more prevalent in women than in men. Also, the association between estrogen status, alveolar bone density, and history of periodontitis in postmenopausal women has been studied (Payne et al., J. Periodontol., 6:24-31 (1997)).
Larger cross-sectional studies, as well as longitudinal and mechanism studies, are needed to better define the relationship between osteoporosis, osteopenia, and oral bone loss, periodontal disease, and tooth loss. The role of factors involved in the regulation of bone mineral density in men as well as in postmenopausal women needs to be evaluated further with reference to oral bone loss, tooth loss, and periodontal disease. Variables such as sex, race, dietary calcium and phosphorus, vitamin D intake, exercise, body mass index, smoking, genetics, medication use, reproductive history, and psychosocial factors need to be assessed in depth. In addition, reliable and valid criteria and imaging technologies for assessing osteoporosis and oral bone loss are needed to better elucidate the full relationship between skeletal and mandibular bone mineral density, periodontal disease, alveolar ridge resorption, and tooth loss.
II. Salivary Mucins
The functional properties of saliva proteins, known as salivary mucins, relative to oral health status are the subject of continuing research (Ayad, et al., J. Dent. Res., 79:976-982 (2000)). The existence of high-molecular-weight glycoproteins in saliva and saliva secretions, called mucins, has been recognized for nearly thirty years (Offner, et al., Adv. Dent. Res., 14:69-75 (2000)). Mucins are essential for oral health and perform many diverse functions in the oral cavity. For example, mucins are the principal protein components of the mucous layer which coats epithelial surfaces in the gastrointestinal, respiratory, and reproductive tracts. This layer forms a viscous barrier which protects the underlying epithelium from desiccation, mechanical injury, and microbial assault, while allowing for active absorption and secretion by mucosal cells. Mucins are also secreted by salivary glands and are thought to have a major role in the protection of oral epithelial surfaces, as well as in the non-immune host defense system in the oral cavity (Offner, et al., supra).
From a biochemical standpoint, mucins are comprised of approximately 15%-20% protein, and up to 80% carbohydrate, present largely in the form of O-linked glycans (Strous and Dekker, Crit. Rev. Biochem. Mol. Biol., 27:57-92 (1992); Gendler and Spicer, Ann. Rev. Physiol., 57:607-634 (1995)). Serine and threonine are the most abundant amino acids and serve as the attachment sites for these carbohydrate chains. Many mucins have monomeric molecular weights greater than two million Daltons, and form multimers more than ten times that size (Offner et al., supra). To date, eleven distinct human mucin genes have been isolated and have been numbered MUC1-MUC4, MUC5AC, MUC5B, MUC6-8, and MUC11-MUC12, in the order of their discovery.
These mucins share several common properties. The polypeptide backbone can be divided into three regions. The central region is enriched in serine, threonine, and sometimes proline, and contains tandemly repeated sequences ranging in length from 8 to 169 amino acids. This domain serves as the attachment site for the O-glycans, and each mucin has a unique, signature tandem-repeat sequence. The N- and C-terminal regions of mucins are non- or sparsely glycosylated with both O- and N-linked sugars. In many mucins, these flanking regions are cysteine-rich, containing nearly 10% cysteine. Mucins could be organized into three distinct classes: the large gel-forming mucins (i.e., MUC2, MUC5AC, MUC5B, and MUC6); the large membrane-associated mucins (i.e., MUC1, MUC3, MUC4, and MUC12); and the small soluble mucins represented by MUC7. Insufficient information is available to assign MUC8 and MUC 11 to one of these categories (Ofnner, et al., supra).
The MUC7 gene has previously been reported (Bobek, et al., Genomics, 31:277-282 (1998)). The MUC7 mucin is generally regarded as having the ability to bind to and aggregate several species of oral bacteria, including several strains of S. mutans, and A. actinomycetemcomitans. The former is thought to be the most cariogenic of the oral bacteria and the latter is one of two major pathogens in periodontal disease. The MUC7 mucin also binds C. albicans and can have candidicidal activity. Desialylation of the mucin apparently destroys its ability to aggregate some species of oral bacteria. Recent studies further indicate that MUC7 mucin binds oral neutrophils on a different oligosaccharide motif than is used to bind oral bacteria. With regard to the primary site of binding to oral bacteria, recent studies suggest that a non-glycosylated domain of MUC7 mucin can be more responsible than its oliogosaccharides.
Bolscher et al., (J. Dent. Res., 78:1362-1369 (1999)) have developed an ELISA for MUC7 that quantitated mucin in stimulated separate salivas from the parotid, submandibular, and sublingual glands of six subjects. The Bolscher study reported the mean concentration of MUC7 for each of the three glandular salivas. Rayment et al. (J. Dent. Res., (2000) 19:1765-1772) have developed a capture ELISA for MUC7 and measured the mean concentration in the stimulated whole saliva of 61 subjects.
In addition, others have either studied the functional properties of MUC7 or the concentrations of other saliva proteins relative to oral health status. Prakobphol, et al. have studied the different types of oligosaccharide chains on MUC7 (Biochemistry 38:6817-6825 (1999)). Prakobphol, et al. reported that different individuals have different classes of oligosaccharides on their MUC7, but they did not include a population study, nor did they quantitate the mucin or its oligosaccharides. Bobek, et al. first cloned the MUC7 gene and is now studying its functional properties (Genomics 31:277-282 (1998)). Ayad, et al., have systematically tested the relationship of various components in saliva to oral health status (J. Dent. Res. 79:976-982 (2000)).
U.S. Patent Publication No. 2003/0040009 A1 to Denny et al., which is incorporated herein by reference, describes the relationship of mucin concentration to DFT (decayed and filled permanent teeth). The mucin test, as described in U.S. Patent Publication No. 2003/0040009 A1, comprises first separating a salivary mucin, e.g., MUC7 mucin, from all other sialic acid-containing molecules in the saliva, by known methods such as sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sialic acid attached to the mucin is then quantitated and reported.
There have been many attempts to measure other factors in saliva and then relate such factors to the caries experience of the individual donors. However, the vast majority of studies, which have reported values for pH, various ions, macromolecules, and flow rate, have found little evidence of a correlation. A few studies have shown a small amount of correlation, though without consensus between studies. Thus it would be of value to develop a test that exhibits a strong correlation between a specific factor and the risk level for caries development, and would allow for the prediction or forecasting of existing caries levels. In addition, this test should be simple and accurate.
Despite advances, caries remains a disease experienced by 85% of U.S. school children before the age of 17. Though wide spread, approximately 75% of all caries is concentrated in only 25% of the population (U.S. Dept. of Health & Human Services. National Institutes of Health Consensus Development Conference Statement. Diagnosis and Management of Dental Caries Throughout Life (2001)). At present, these caries-prone individuals are identified by accumulated caries experience, usually in an office setting. However, this professional judgment is based on personal anecdotal experience. Literature on the prediction of future caries development in specific individual subjects is lacking. The current level of scientific effort is reflected in 24 studies of the efficacy of caries preventives and non-invasive treatments, whose subject populations were selected on the basis of “high-risk” or “caries-active.” These studies are summarized in “Evidence Report/Technology Assessment, Number 36: Diagnosis and Management of Dental Caries” (U.S. Dept. of Health & Human Services. National Institutes of Health Consensus Development Conference Statement Diagnosis and Management of Dental Caries Throughout Life (2001)). With average ages ranging from 1 to 13 years, these studies averaged 175 individuals per study. Each study was evaluated on the quality of the evidence for judging the efficacy of a particular treatment. The four grades achievable were “good,” “fair,” “poor,” and “incomplete.” Only four of the individual studies received a fair rating for their results and conclusions. The remaining 20 were rated as incomplete. While a number of factors were at play in this rating, special note was made of the lack of consistency in the inclusion criteria for “at-risk” and “caries-active” participants.
In general, these studies have used either Streptococcus mutans titers or past caries history, such as dmft or dmfs (deciduous teeth), and DMFT or DMFS (permanent teeth), to select the high risk and/or caries-active subjects. A problem associated with these inclusion criteria is the subjectiveness associated with the actual diagnoses, as well as lack of agreement on what number constitutes a caries-prone individual. This uncertainty about identification of individuals who have a high potential for future caries development is exemplified in a divided study that was partitioned into “high-risk” (S. mutans titer-based), and “caries-active” (DMFS-based) subjects. The same treatment agent gave a 33% reduction in new caries in the “high-risk” group and a 9% increase in the “caries-active” group (U.S. Dept. of Health & Human Services. National Institutes of Health Consensus Development Conference Statement. Diagnosis and Management of Dental Caries Throughout Life (2001)). In addition to the confusion about what inclusion criteria best identify those with the highest projected rates of cariogenesis, the assessment report notes that neither approach to caries risk assessment has ever been validated.
Thus, there is value and need for a test that provides standard subject-identification criteria and that leads to a consistent diagnosis throughout childhood and early adulthood. A preferred test would have a uniform design such that it would accommodate all age groups and races.